The overall goal of this proposal is to develop a human-friendly and less-expensive method of prophylaxis for respiratory syncytial virus (RSV) infection and other respiratory viruses using intranasal administration of chitosan nanoparticles containing pDNA, which allows de novo expression of IFNs. Forty-five years after its discovery, no effective vaccine or treatment is currently available against RSV infection. Current prophylaxis for RSV comprises a monthly treatment with a humanized antibody to RSV fusion protein (Palivizumab, Synagis TM) that is only moderately effective in high risk infants, more expensive and its application to adults and elderly is unclear. This proposal focuses on a new concept for prophylaxis and is based on the discovery that intranasal administration of plasmid DNA(s) (pDNA) encoding IFN-gamma, (plFN-gamma) results in a significant decrease of RSV infection in a mouse model. Also, exogenous IFN-gamma/ prevents RSV infection of human epithelial cells and significantly decreases established infection. Also, a combination of plFN-gamma and plFN-beta induces more significant decrease in RSV infection of epithelial cells than either of them alone. Chitosan naoparticles have been developed that facilitate IFNgamma gene transfer in mice. The Transgenex Therapeutics (TGT), a start-up biotech company is developing biopolymers, called chlipoplexes, which protect the pDNAs and target them to the epithelial cells, macrophages or dendritic cells (DCs), where the virus replicates. Together, these developments have led to the working hypothesis that chlipoplexes will deliver effectively the pDNAs without any significant adverse effects in the nasal mucosa and provide significant protection against viral infections. These hypotheses will be tested in this proposal under the following specific aims. Aim #1. To screen and identify the most effective plFN(s) for RSV infection. It is planned to develop human-friendly, nonintegrating pDNA vectors for various IFNs, either singly or in combination and test their antiviral properties in human cell lines. Aim #2. To develop and test chlipoplexes for optimized delivery and expression of plFNs. It is planned to develop chlipoplexes containing pIFNs (CLIP) and test their effectiveness to attenuate RSV infection in the three dimensional air-liquid interphase (ALl) cultures, which mimics the nasal mucosa of humans. Aim #3. To test the selected CLIP(s) in mice. It is planned to test the selected top two CLIPs using the mouse model of RSV infection and investigate their antiviral activity in relation to dosage, safety, and immunological effects in mice. It is anticipated that the results of the studies proposed herein will enhance our understanding of the potential of CLIP as agents for prophylaxis against RSV infection and will move the promising CLIP(s) into phase I clinical trials.